Simultaneous determination of Phenylalanine, Tyrosine and Tryptophan

by HPLC-UV

Hicham SAM a,*,c; Soufiane EL ASSRI a,c; Assmae AMEZIAN b; Chaymae ROCHDI c, Mohammed CHOUKRI a,b,c
a Laboratory of Biochemistry and Biotechnology, Faculty of Sciences of Oujda, Mohammed First
University, PB 717, 60000 Oujda, Morocco
b Faculty of Medicine and Pharmacy of Oujda, Mohammed First University, PB 4867, 60049 Oujda, Morocco
c Central Laboratory, Mohammed VI University Hospital, PB 4806, 60049 Oujda, Morocco

Email de correspondance : hichamsam233@gmail.com

Introduction:
Phenylalanine (Phe) and tyrosine (Tyr) are the most reliable indicators for the diagnosis of phenylketonuria (PKU). Tryptophan metabolism is
disturbed in mental depression, and the induction of indoleamine 2,3-dioxygenase increases kynurenine production. ). The purpose of this study
is to establish a simple and rapid method for the determination of Phe, Tyr and Trp in serum by high performance liquid chromatography
(HPLC).

PROBLÉMATIQUE & OBJECTIFS

They are complicated and expensive methods when they are not
applied worldwide. Thus, there is no protocol for the simultaneous
determination of the three markers from which development is
indispensable for better separation and quantification. Our work
allows the development of a simple method without derivation and
inexpensive for the simultaneous determination of the three amino
RESULTATS & DISCUSSION
acids in the blood and therefore for systematic screening neonatal of
the three diseases

MATERIELS ET METHODES

Serum samples were collected and 40 μL of each was transferred to

Eppendorf tube. An equal volume of 5% (V/V) perchloric acid was
added and mixed thoroughly. The mixtures were centrifuged at
13000 g for 10 min at 4 °C. Then 20 μL of supernatants were
injected into HPLC system for analysis. Chromatography was carried
out by isocratic elution on a VDS Optilab column (150 × 4.6 mm; 5
μm particle size C18). The mobile phase consisted of 4% acetonitrile
and 1% methanol in water (v/v). Flow rate was 1 mL/min. The eluate
was monitored by the ultraviolet detector set at 214 nm.

CONSCLUSION Fig 1 : HPLC chromatograms of Tyr, Phe and Trp

In conclusion, we have successfully established a HPLC-UV method
for simultaneously detecting Phe, Tyr and Trp levels in serum (Fig. 1).
The development of this method encourages us to continue our
investigations in order to validate simple methods for the
determination of other amino acids.
This work has made it possible to optimize sample preparation
in all three directions: Complexity, time and cost. Unlike the
HPLC method with derivation which requires complicated
manipulations with expensive products, our method is simple
inexpensive and uncomplicated.

REFERENCES BIBLIOGRAPHIQUE
[1] Smith I, Cook B, Beasley M. Review of neonatal screening programme for phenylketonuria. BMJ 1991;303:333–5.
[2] Miller JB, Shapira E. The advantage of phenylalanine to tyrosine ratio for the early detection of phenylketonuria. Clin Chim Acta
1998;270:177–81
[2] M. Yust, J.Pedroche, J. Girón-Calle, J. Vioque, F. Millán, M. Alaiz, Determination of tryptophan by high-performance liquid chromatography
of alkaline hydrolysates with spectrophotometric detection, Food Chem. 85 (2004) 317–320.